Antibody-mediated neutralization is widely accepted to be a major mechanism by which SIV and HIV-1 infection can be aborted. However, the mechanism(s) of this neutralization process in vivo is unknown, and current in vitro assays are insensitive indicators of in vivo virus neutralization. To address this problem, our lab has begun to develop more sensitive neutralization assays to help dissect the early events of virus infection involved in antibody-mediated neutralization (Le., virus binding to the host cell, virus-host cell fusion and virus internalization). For these assays, we will use a unique panel of rhesus monoclonal antibodies derived from monkeys infected with SIV that are divided equally between those that neutralize efficiently and those that fail to demonstrate neutralization in vitro. Our first goal is to define the mechanism of neutralization mediated by these antibodies so that we may identify selected mechanisms to determine the correlation between in vitro neutralization assays and in vivo virus neutralization. Second, we will determine the effects of sequence variation on specific mechanisms of antibody-mediated neutralization. For these studies we will follow the evolution of viral variants that emerge in vivo with the goal of identifying specific changes in the viral envelope that are associated with the transition from a neutralization sensitive to a neutralization insensitive phenotype in vivo. In addition, we will utilize viruses with defined modifications in the envelope (i.e., selected point mutations defined by sequence analyses of viral variants that emerge in vivo, deletions of N-linked glycosylation sites, mutations or deletions within defined variable and/or conserved regions) to correlate mechanisms of virus neutralization with envelope variation. In this manner, we will provide evidence for the specificity and mechanism of antibody-mediated neutralization in vitro, and identify specific genotypic changes that are associated with functional changes in virus neutralization in vivo. These studies will provide a framework to define the role of type-specific and broadly neutralizing antibody in controlling virus infection and disease in the SIV/rhesus macaque model in vivo, a future goal of this laboratory.